The present invention relates to novel K99-5041 substance having inhibitory action for lipid metabolism and a process for production thereof.
Prior Art
Sterol such as cholesterol in human or ergosterol in fungi is biosynthesized. Enzymes involved in the sterol biosynthesis are recognized as the targets for development of preventive and therapeutic agents for human hyperlipidemia or arteriosclerosis and development of antifungal agents (Tomoda, H. and Omura, S.: Enzyme Technology for Pharmaceutical and Biological Applications, Ed. Kirst, A. et al., Chapter 15, pp. 343-378, Marcel Dekker, Inc. NY, 2001).
Statin series compounds represented by pravastatin specifically inhibit hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase, a one of rate-limiting enzyme of cholesterol biosynthesis, and reduce cholesterol level in blood, and are used as preventive and therapeutic drugs for arteriosclerosis in clinical practice. However, in case of statin series compounds used at present, there is a possibility to inhibit biosynthesis of non-sterol compounds which are essential components in vivo. Consequently, enzymes involved in the downstream from squalene which is an intermediate in the biosynthesis are expected as superior target for drugs.
Azole series compounds used for treatment of mycosis such as 2,4-difluoro-xcex1,xcex1-bis-(1H,1,2,4-triazole-1-ylmethyl)benzyl alcohol (generic name: fluconazole, I.C.N. Pharmaceuticals Inc., U.S.A.) and 1-[2-(2,4-dichlorobenzyloxy)-2-(2,4-dichlorophenyl)ethyl]imidazole (generic name: miconazole, Sigma Inc., U.S.A.) are used in practice as antifungal agents which inhibit C-14 demethylation of ergosterol. However, appearance of drug resistant microorganisms caused by long-term or repetitive administration of azole series antifungal agents becomes problem. Consequently, development of drugs with high safety and low incidence of resistant strains is urgently necessary.
Lanosterol synthase is an enzyme generating lanosterol by cyclization of 2,3-oxidosqualene as a substrate and drugs which inhibit such enzyme has not been utilized. Consequently, discovery of pharmaceutical agents which inhibit such the enzyme is thought to solve above problems and are expected to be used in clinical practice as preventive and therapeutic agents for myocardial infarction and cerebral apoplexy caused by hyperlipidemia and arteriosclerosis, or new antifungal agents.
An object of the present invention is to provide novel K99-5041 substance, which is used for clinical practice as preventive or therapeutic agents for diseases caused by accumulation of cholesterol in humans by inhibitory action against lanosterol synthase, and process for production thereof.
We have studied on metabolites produced by microorganisms, and found that substances having inhibitory activity for lanosterol synthase were produced in a culture mass of a microbial strain K99-5041 which was newly isolated from soil. Subsequently, we have isolated and purified the active substance inhibiting lanosterol synthase from the cultured mass, and found substances having chemical structure represented by the formula [I] and [II] hereinbelow. Since these substances have not been known, the substances are designated as K99-5041-C1x substance and K99-5041-C2x substance, and totally designated as K99-5041 substance.
The present invention has been completed based on the above knowledge. An object of the present invention is to provide K99-5041-C1x substance represented by the following formula [I]: 
Another object of the present invention is to provide K99-5041-C2x substance represented by the following formula [II]: 
Further object of the present invention is to provide a composition of novel K99-5041 substance comprising especially K99-5041-C1x substance represented by the following formula [I]: 
and especially K99-5041-C2x substance represented by the following formula [II]: 
More further object of the present invention is to provide a process for production of K99-5041-C1x substance comprising culturing a microorganism belonging to genus Streptomyces and having ability to produce K99-5041-C1x substance in a medium, accumulating K99-5041-C1x substance in a culture fluid and isolating K99-5041-C1x substance from the cultured mass.
Still further object of the present invention is to provide a process for production of K99-5041-C2x substance comprising culturing a microorganism belonging to genus Streptomyces and having ability to produce K99-5041-C2x substance in a medium, accumulating K99-5041-C2x substance in a culture fluid and isolating K99-5041-C2x substance from the cultured mass.
Still more further object of the present invention is to provide a process for production of a composition of K99-5041 substance comprising culturing a microorganism belonging to genus Streptomyces having ability to produce K99-5041-C1x substance and/or K99-5041-C2x substance in a medium, accumulating K99-5041-C1x substance and/or K99-5041-C2x substance in a culture fluid and isolating K99-5041-C1x substance and/or K99-5041-C2x substance from the cultured mass.
Further object of the present invention is to provide a process for production of K99-5041-C1x substance and/or K99-5041-C2x substance wherein a microorganism belonging to genus Streptomyces and having ability to produce K99-5041-C1x substance and/or K99-5041-C2x substance is Streptomyces sp. K99-5041 FERM BP-8272.
Further object of the present invention is to provide a microorganism of Streptomyces sp. K99-5041 FERM BP-8272.
Further object of the present invention is to provide K99-5041-C1x substance, K99-5041-C2x substance or a composition of K99-5041-C1x substance and/or K99-5041-C2x substance for use as a medicament.
Further object of the present invention is to provide K99-5041-C1x substance, K99-5041-C2x substance or a composition of K99-5041-C1x substance and/or K99-5041-C2x substance used for inhibiting lanosterol synthase which synthesizes lanosterol generated by cyclization of 2,3-oxidosqualene as a substrate.
Further object of the present invention is to provide K99-5041-C1x substance, K99-5041-C2x substance or a composition of K99-5041-C1x substance and/or K99-5041-C2x substance used for preventing or treating diseases of myocardial infarction or cerebral apoplexy based on hyperlipidemia and arteriosclerosis caused by accumulation of cholesterol in humans.
Further object of the present invention is to provide K99-5041-C1x substance, K99-5041-C2x substance or a composition of K99-5041-C1x substance and/or K99-5041-C2x substance used for manufacture of drug preparations for inhibiting myocardial infarction, cerebral apoplexy or mycosis.
Further object of the present invention is to provide K99-5041-C1x substance, K99-5041-C2x substance or a composition of K99-5041-C1x substance and/or K99-5041-C2x substance used for prevention or treatment of diseases including myocardial infarction, cerebral apoplexy or mycosis.
The microorganism having ability to produce K99-5041-C1x substance represented by the above formula [I] and K99-5041-C2x substance represented by the above formula [II] or a composition thereof (hereinafter designates as xe2x80x9cK99-5041 substance producing microorganismxe2x80x9d) belongs to genus Streptomyces, and, for example, a strain Streptomyces sp K99-5041, which was isolated by us, is an example of the strain used most effectively in the present invention.
Taxonomical properties of the strain K99-5041 are as follows.
1. Morphological Properties
Vegetative mycelia grow well on various agar media and no fragmentation is observed. Aerial mycelia are abundantly grown on yeast-malt extract agar medium and show white to grayish white color. On microscopic observation, chains of more than 20 spores were observed on the aerial mycelia, and the morphological form is flexuous and size of spore is about 0.9-1.1xc3x970.6-0.7 xcexcm with cylindrical form. Surface of the spore is smooth. No sclerotia, sporangia and zoospore are observed.
2. Properties on Various Media
Culture properties of the producing strain of the present invention determined by the method of E. B Shirling and D. Gottlieb (International Journal of Systematic Bacteriology, 16: 313, 1966) are shown in the following.
Color tone was determined referring to Color Harmony Manual, 4th Ed. (Container Corporation of America, Chicago, 1958) as a standard color, and color name as well as attached code number in the parenthesis.
Unless otherwise noted, results are observation of cultures at 27xc2x0 C. for 2 weeks on various media.
3. Physiological Properties
(1) Formation of melanin pigment
(a) Tyrosine agar negative
(b) Peptone-yeast-iron agar medium negative
(c) Tryptone-yeast liquid negative
(d) Simple gelatin medium (21-23xc2x0 C.) negative
(2) Nitrate reduction negative
(3) Liquefaction of gelatin (21-23xc2x0 C.) (simple gelatin medium) negative
(4) Starch hydrolysis negative
(5) Coagulation of defatted milk (27xc2x0 C.) negative
(6) Peptonization of defatted milk (27xc2x0 C.) negative
(7) Growth temperature 12-33xc2x0 C.
(8) Utilization of carbon sources (Pridham-Gottlieb agar medium)
Utilize: D-glucose, myo-inositol
Slightly utilize: D-fructose
Not utilize: L-arabinose, D-xylose, D-mannitol, raffinose L-rhamnose, sucrose, melibiose
(9) Decomposition of cellulose negative
4. Composition of Cell Wall
2,6-diaminopimelic acid of cell wall is LL type. Main menaquinones are MK-9 (H6) and MK-9 (H8).
5. Conclusion
Taxonomical properties of the strain of the present invention are summarized as follows. 2,6-Diaminopimelic acid in the cell wall is LL type and main menaquinones are MK-9 (H6) and MK-9 (H8). Morphology of the spore chain is flexuous, forming with long spore chains and smooth spore surface. Various properties on the culture are exhibiting pale yellow color tone with white to grayish aerial mycelia. No production of melanin pigment is observed, but yellow soluble pigments formation was observed.
The present strain exhibiting the above morphological properties, culture properties and physiological properties was identified as the strain belonging to genus Streptomyces, and was thought to be the strain belonging to the gray series in the classification by Pridham and Tresner (Bergey""s Manual of Determinative Bacteriology, 8th Ed., page 748-829, 1974). The strain was deposited as Streptomyces sp. K99-5041 in International Patent Organism Depository, National Institute of Advanced Industrial Science and Technology, AIST Tsukuba Central 6, 1-1, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, 305-8566 Japan on Mar. 14, 2002 as permanent depository number FERM P-18765. Thereafter, request for transfer of the strain to the international depository was accepted on Jan. 9, 2003, under the Budapest Treaty relating to international approval for deposition of microorganisms on the patent procedure in the International Patent Organism Depository, National Institute of Advanced Industrial Science and Technology, AIST Tsukuba Central 6, 1-1, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, 305-8566 Japan. The permanent depository number is FERM BP-8272.
K99-5041 substance producing strain as the preferable strain used in the present invention is explained. However, since the morphological properties of microorganisms are generally very easily mutated and are not constant. Natural mutation or artificial mutation generally performed by ultraviolet irradiation or chemical mutagens such as N-methyl-Nxe2x80x2-nitro-N-nitrosoguanidine and ethyl methansulfonate, are well known. The strain belonging to genus Streptomyces and having ability to produce K99-5041 substance, including the artificial mutants as well as natural mutants, can be used in the present invention. In addition, all strains belonging to genus Streptomyces and having ability to produce K99-5041 substance including strains mutated by cell engineering technology such as cell fusion, gene manipulation, etc., can be used in the present invention.
In production of K99-5041-C1x substance and/or K99-5041-C2x substance of the present invention, at first, K99-5041 substance producing strain belonging to genus Streptomyces is cultured in a preferable medium. In the culture of the strain, conventional medium for culturing fungi was generally applied. Medium containing assimilable carbon sources for microorganism, digestible nitrogen sources and, if necessary, inorganic salts can be used as nutrient medium.
Examples of assimilable carbon sources are glucose, sucrose, molasses, starch, dextrin, cellulose, glycerin, organic acid, etc. are used independent or in combination. Examples of digestible nitrogen sources are organic nitrogen sources such as peptone, meat extract, yeast extract, dry yeast, soybean powder, corn steep liquor, cotton seed powder, casein, soybean protein hydrolyzate, amino acids and urea, and inorganic nitrogen sources such as nitrates and ammonium salts are used independent or in combination.
If necessary, inorganic salts or heavy metallic salts such as sodium, potassium, calcium, magnesium, phosphate, etc. are added. Further, if necessary, trace nutrients, growth promoting agents and precursors preferable for production of K99-5041 substance are optionally added.
Culture is preferably performed by aerobic condition such as conventional shake culture or aeration agitation culture. For industrial culture, submerged aeration agitating culture is preferable. The pH of the medium is preferably performed at neutral condition. Culturing temperature can be performed at 20-37xc2x0 C., generally at 24-30xc2x0 C., preferably at 27xc2x0 C. In case of liquid culture, when culture is performed generally for 3-6 days, K99-5041 substance of the present invention is produced and accumulated, and when the amount of accumulation reaches maximum in the culture medium, the cultivation may preferably be terminated.
A condition of culture such as composition of culture, properties of liquid culture medium, culturing temperature, agitation rate and amount of aeration may naturally be adjusted and selected in order to obtain preferable result of cultivation depending on types of strain and external condition. In the liquid culture, if foaming occurs, antifoam agents such as silicone oil, vegetable oil and surface active agent can preferably be used.
Since K99-5041 substance accumulated in the thus obtained cultured broth is contained in the cultured filtrate or cultured mycelia, the cultured broth is filtered with optionally adding filter aid such as Celite, Hyflosupercell, etc., or is centrifuged to separate into the cultured filtrate and the mycelia, then organic solvent extracts from the cultured filtrate and mycelia are concentrated and K99-5041 substance is preferably collected therefrom.
Isolation of K99-5041 substance from the cultured filtrate can be performed by extracting the cultured filtrate with water immiscible organic solvent such as ethyl acetate, butyl acetate and benzene, and concentrating the extract in vacuo to obtain crude K99-5041 substance. The crude substance is treated by conventional known method used for purification of fat-soluble substances, for example, column chromatography using carrier such as silica gel and alumina to isolate and purify K99-5041 substance.
In order to isolate K99-5041 substance from mycelia, the mycelia are extracted by aqueous water miscible organic solvent such as acetone and aqueous methanol. The obtained extract is concentrated in vacuo, and the concentrated material is extracted with water immiscible organic solvent such as ethyl acetate, butyl acetate and benzene, then the thus obtained extract is combined with the extract obtained from the above culture liquid and is treated for separation and purification of K99-5041 substance. Further, K99-5041 substance can be isolated and purified by the same method described hereinabove.
Physicochemical properties of K99-5041-C1x substance and K99-5041-C2x substance of the present invention are described as follows.
[I] K99-5041-C1x substance
(1) Molecular formula: C22H41N [High resolution FAB mass spectrometry: m/z 319.3234 (M)] (Calculated: 319.3239)
(2) Molecular weight: 319 [EI mass spectrometry: m/z 319 (M)+]
(3) Specific rotation: [xcex1]D24=xe2x88x923.2xc2x0 (c=0.26, methanol)
(4) Ultraviolet absorption spectrum (in methanol): as shown in FIG. 1, maximum absorption at 235 nm (log xcex5=14,500)
(5) Infrared absorption spectrum (KBr Tablet): as shown in FIG. 2, xcexmaxKBr cmxe2x88x921: 3435, 2924, 2854, 1606, 1466, 1385
(6) Solubility in solvent: soluble in methanol, ethanol, acetonitrile, ethyl acetate, chloroform and dimethyl sulfoxide, and insoluble in water.
(7) Grouping for acidic, neutral and basic: Weak basic substance.
(8) Color and form of substance: pale yellow oily substance.
(9) Proton nuclear magnetic resonance spectrum: Proton nuclear magnetic resonance spectrum measured by using JEOL, nuclear magnetic resonance spectrometer (in deuteriochloroform, 500 MHz) is as shown in FIG. 3. Chemical shifts (ppm) are as follows. 0.86 (6H), 1.15 (2H), 1.2-1.4 (20H), 1.43 (2H), 1.51 (1H), 2.06 (3H), 2.10 (2H), 2.48 (2H), 3.86 (2H), 5.75 (1H).
(10) Carbon nuclear magnetic resonance spectrum: Carbon nuclear magnetic resonance spectrum measured by using JEOL, nuclear magnetic resonance spectrometer (in deuteriochloroform, 125.65 MHz) is as shown in FIG. 4. Chemical shifts (ppm) are as follows. 15.9 (1C), 22.7 (2C), 26.8 (1C), 27.4 (1C), 28.0 (1C), 29.0 (1C), 29.4-30.0 (9C), 30.5 (1C), 39.1 (1C), 57.8 (1C), 126.3 (1C), 143.2 (1C), 171.7 (1C).
As described hereinabove, as the results of examining various physicochemical properties and spectral data of K99-5041-C1x substance, K99-5041-C1x substance was determined as having chemical structure represented by the following formula [I]. 
[II] K99-5041-C2x substance
(1) Molecular formula: C22H41N [High resolution FAB mass spectrometry: m/z 319.3224 (M+)] (Calculated: 319.3239)
(2) Molecular weight: 319 [EI mass spectrometry: m/z 319 (M)+]
(3) Specific rotation: [xcex1]D24=xe2x88x921.2xc2x0 (c=0.17, methanol)
(4) Ultraviolet absorption spectrum (in methanol): as shown in FIG. 5, maximum absorption at 235 nm (log xcex5=13,900)
(5) Infrared absorption spectrum (KBr Tablet): as shown in FIG. 6, xcexmaxKBr cmxe2x88x921: 3431, 2924, 2852, 1606, 1466, 1385, 1261, 1099, 1032, 804.
(6) Solubility in solvent: soluble in methanol, ethanol, acetonitrile, ethyl acetate, chloroform and dimethyl sulfoxide, and insoluble in water.
(7) Grouping for acidic, neutral and basic: Weak basic substance.
(8) Color and form of substance: white oily substance.
(9) Proton nuclear magnetic resonance spectrum: Proton nuclear magnetic resonance spectrum measured by using JEOL, nuclear magnetic resonance spectrometer (in deuteriochloroform, 500 MHz) is as shown in FIG. 7. Chemical shifts (ppm) are as follows. 0.88 (3H), 1.2-1.4 (26H), 1.43 (2H), 2.06 (3H), 2.10 (2H), 2.48 (2H), 3.86 (2H), 5.75 (1H).
(10) Carbon nuclear magnetic resonance spectrum: Carbon nuclear magnetic resonance spectrum measured by using JEOL, nuclear magnetic resonance spectrometer (in deuteriochloroform, 125.65 MHz) is as shown in FIG. 8. Chemical shifts (ppm) are as follows. 14.1 (1C), 15.9 (1C), 22.7 (1C), 26.8 (1C), 29.0 (1C), 29.4-29.7 (11C), 30.5 (1C), 31.9 (1C), 57.8 (1C), 126.2 (1C), 143.2 (1C), 171.7 (1C).
As described hereinabove, as the results of examining various physicochemical properties and spectral data of K99-5041-C2x substance, K99-5041-C2x substance was determined as having chemical structure represented by the following formula [II]
Various physicochemical properties of K99-5041-C1x substance and K99-5041-C2x substance were described hereinabove in detail. Compounds having identical properties have not been reported, and K99-5041-C1x substance and K99-5041-C2x substance are determined as novel substances.
Biological properties of K99-5041 substance of the present invention are described hereinbelow.
(1) Inhibitory Action Against Lanosterol Synthase of Human Origin
Lanosterol synthase activity was assayed by partially modified method of Kusano et al (Chem. Pharm. Bull. 39, 239-241, 1991). Enzyme origin used is as follows. Lanosterol synthase cDNA derived from human origin (Sung et al. Biol. Pharm. Bull., 18, 1459-1461, 1995) was expressed by glyceraldehydes-3-phosphate dehydrogenase promoter in lanosterol synthase deficient yeast GIL77 (Kushiro et al., Eur. J. Biochem., 256, 238-241, 1998). The thus obtained cell-free extract derived from transformed yeast was used.
The above transformed yeast was homogenized in buffer A [0.1 M potassium-phosphate buffer solution (pH 7.4), 0.45 M sucrose, 1 mM EDTA and 1 mM dithiothreitol] in the presence of acid treated glass beads using Waring blender [Type 500ACD, Sakuma Seisakusho Co., Japan]. Supernatant obtained by centrifugation, 8600xc3x97g, was prepared to make protein concentration of 10 mg/ml by adding the buffer A hereinabove to prepare the cell-free extract.
A substrate, [14C](3S)-2,3-oxidosqualene was prepared biosynthetically by culturing lanosterol deficient yeast GL7 (Gollub et al. J. Biol. Chem. 252, 2846-2854, 1977) in the presence of [1-14C]sodium acetate to incorporate the radioactivity into oxidosqualene.
Assay of lanosterol synthase was performed as follows. [14C](3S)-2,3-oxidosqualene (final concentration, 0.17-0.42 xcexcM, 4.5 nCi) and K99-5041 substance were added in buffer B [0.1 M potassium-phosphate buffer (pH 7.4) and 0.1% Triton X-100] to prepare total volume 900 xcexcl, preincubated at 37xc2x0 C. for 10 minutes, added enzyme 1 mg protein to prepare total 1 ml, and incubated at 37xc2x0 C. for 60 minutes.
Subsequently, 6% potassium hydroxide/ethanol was added thereto to terminate the reaction, and the mixture was saponificated by incubating at 37xc2x0 C. for 10 minutes, added cyclohexane 2 ml and stirred well. The cyclohexane layer 1.6 ml was dried up, spotted on TLC plate [silica gel plate (with concentration zone), Merck Inc., U.S.A., thickness 0.25 mm: Art. No. 11798] and developed with solvent of benzene/acetone (19:1, v/v). Next, amounts of unreacted [14C](3S)-2,3-oxidosqualene and generated [14C]lanosterol were quantitatively assayed using the photo image analyzer BAS-1500 (Fuji Photo Film Co., Japan) to calculate inhibition rate of lanosterol synthesis.
As the results, 50% inhibition concentration (IC50) for lanosterol synthase activity of K99-5041 substance was determined as 15 xcexcM for K99-5041-C1x substance and 18 xcexcM for K99-5041-C2x substance.
As explained in detail, since K99-5041 substance of the present invention exhibits inhibitory action against lanosterol synthase, it is expected to be useful for preventive and therapeutic agents for disease caused by cholesterol accumulation in humans and antifungal agents.